The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes

P-selectin is a cell adhesion molecule required transiently on the surface of activated platelets and endothelial cells as a receptor for leukocytes. It is stored in secretory granules in platelets, endothelial cells, and transfected neuroendocrine cells and is rapidly delivered to the plasma membrane upon exocytosis of the secretory granules. It is then rapidly internalized in endothelial cells and transfected cells. We find that in transfected neuroendocrine PC12 cells, the fraction of P-selectin that is not targeted to secretory granules is rapidly degraded. In transfected CHO fibroblasts, which lack secretory granules, P-selectin was degraded with a half time of 2.3 h in plated cells, while low density lipoprotein receptor (LDL-R) had a half life of 9 h. In cells cultured in ammonium chloride to inhibit lysosomal proteinases, P-selectin was protected from degradation and rapidly accumulated in vesicles enriched in lgp-B, a resident lysosomal membrane protein. The cytoplasmic domain of P- selectin was sufficient to confer rapid turnover on LDL-R. Deletion of 10 amino acids from the cytoplasmic domain of P-selectin extended the half life to 9.5 h and abrogated rapid lysosomal targeting in the presence of ammonium chloride, implicating this sequence as a necessary element of a novel lysosomal targeting signal. The rate limiting step in degradation occurred after internalization from the cell surface, indicating that sorting of P-selectin away from efficiently recycled proteins occurs in endosomes. We propose that this sorting event represents a constitutive equivalent of receptor down regulation, and may function to regulate the expression of P-selectin at the surface of activated endothelial cells.     

Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.     

Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.     

Aftiphilin and gamma-synergin are required for secretagogue sensitivity of Weibel-Palade bodies in endothelial cells

Formation of secretory organelles requires the coupling of cargo selection to targeting into the correct exocytic pathway. Although the assembly of regulated secretory granules is driven in part by selective aggregation and retention of content, we recently reported that adaptor protein-1 (AP-1) recruitment of clathrin is essential to the initial formation of Weibel-Palade bodies (WPBs) at the trans-Golgi network. A selective co-aggregation process might include recruitment of components required for targeting to the regulated secretory pathway. However, we find that acquisition of the regulated secretory phenotype by WPBs in endothelial cells is coupled to but can be separated from formation of the distinctive granule core by ablation of the AP-1 effectors aftiphilin and γ-synergin. Their depletion by small interfering RNA leads to WPBs that fail to respond to secretagogue and release their content in an unregulated manner. We find that these non-responsive WPBs have density, markers of maturation, and highly multimerized von Willebrand factor similar to those of wild-type granules. Thus, by also recruiting aftiphilin/γ-synergin in addition to clathrin, AP-1 coordinates formation of WPBs with their acquisition of a regulated secretory phenotype.     

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.     

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.     

Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules

Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20-25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.     

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.     

A new member of the sorting nexin family interacts with the C-terminus of P-selectin

P-selectin is a cell adhesion molecule found in platelets and endothelial cells mediating binding of leukocytes. It is stored in secretory granules and expressed at the plasma membrane after cell activation. After rapid internalisation P-selectin recycles or is degraded. The 35 amino acid cytoplasmic domain of P-selectin contains signals for sorting into secretory granules, for endocytosis and for delivery to lysosomes. To investigate protein-protein interactions, we performed two-hybrid screening using the cytoplasmic domain of P-selectin as bait. KIAA0064 was identified as a putative intracellular P-selectin binding protein. Because the protein contains a phox homology (PX) domain in the N-terminus which is a characteristic feature of the sorting nexin (SNX) family, it was named SNX17. The PX domain is not required for binding of P-selectin in the two-hybrid system. Expression of a fusion protein between SNX17 and green fluorescent protein demonstrated localisation of SNX17 in the cytosol and to membranes.     

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.     

The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles.

Diversion of membrane proteins from the trans-Golgi network (TGN) or the plasma membrane into the endosomal system occurs via clathrin-coated vesicles (CCVs). These sorting events may require the interaction of cytosolic domain signals with clathrin adaptor proteins (APs) at the TGN (AP-1) or the plasma membrane (AP-2). While tyrosine- and di-leucine-based signals in several proteins mediate endocytosis via cell surface CCVs, segregation into Golgi-derived CCVs has so far only been documented for the mannose 6-phosphate receptors, where it is thought to require a casein kinase II phosphorylation site adjacent to a di-leucine motif. Although recently tyrosine-based signals have also been shown to interact with the mu chain of AP-1 in vitro, it is not clear if these signals also bind intact AP-1 adaptors, nor if they can mediate sorting of proteins into AP-1 CCVs. Here we show that the cytosolic domain of the lysosomal membrane glycoprotein lamp-1 binds AP-1 and AP-2. Furthermore, lamp-1 is present in AP-1-positive vesicles and tubules in the trans-region on the Golgi complex. AP-1 binding as well as localization to AP-1 CCVs require the presence of the functional tyrosine-based lysosomal targeting signal of lamp-1. These results indicate that lamp-1 can exit the TGN in CCVs and that tyrosine signals can mediate these sorting events.     

P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells

P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P-selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P-selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules.     

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.     

Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.     

Cytoplasmic transport signal is involved in phogrin targeting and localization to secretory granules

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic beta-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein (AP)-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules.     

Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.     

Tyrosine-based endocytic motifs stimulate oligomerization of AP-2 adaptor complexes

The clathrin adaptor complex AP-2 functions in the assembly of clathrin-coated vesicles at the plasma membrane where it serves to couple endocytic vesicle formation to the selection of membrane cargo proteins. Recent evidence suggests that binding of tyrosine-based endocytic sorting motifs may induce a conformational change within the AP-2 adaptor complex that could enhance its interaction with other cargo molecules and with the membrane. We report here that soluble tyrosine-based endocytic sorting motif peptides facilitate clathrin/AP-2 recruitment to liposomal membranes and induce adaptor oligomerization even in the absence of a lipid bilayer. These effects are specific for endocytic motifs of the type Yxxphi whereas peptides corresponding to NPxY- or di-leucine-containing sorting signals are ineffective. Our data may help to explain how the highly cooperative assembly of clathrin and adaptors could be linked to the selection of membrane cargo proteins.     

Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ~90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.     

Sorting it out: AP-2 and alternate clathrin adaptors in endocytic cargo selection

The AP-2 adaptor complex is widely viewed as a linchpin molecule in clathrin-mediated endocytosis, simultaneously binding both clathrin and receptors. This dual interaction couples cargo capture with clathrin coat assembly, but it has now been discovered that the association with cargo is tightly regulated. Remarkably, AP-2 is not obligatory for all clathrin-mediated uptake, and several alternate adaptors appear to perform similar sorting and assembly functions at the clathrin bud site.     

Adaptable adaptors for coated vesicles

Adaptors select cargo for inclusion into coated vesicles in the late secretory and endocytic pathways. Although originally there were thought to be just two adaptors, AP-1 and AP-2, it is now clear that there are many more: two additional adaptor complexes, AP-3 and AP-4, which might function independently of clathrin; a family of monomeric adaptors, the GGAs; and an ever-growing number of cargo-specific adaptors. The adaptors are targeted to the appropriate membrane at least in part by interacting with phosphoinositides, and, once on the membrane, they form interconnected networks to get different types of cargo into the same vesicle. Adaptors participate in trafficking pathways shared by all cells, and they are also used to generate specialized organelles and to influence cell fate during development.